Regulation of the immune system

ABSTRACT

The addition of 5-androstene 3(beta),17(beta)diol and/or 5-androstene 3(beta),7(beta),17(beta) triol to growth media increases proliferation of lymphocytes in culture. By methods of the invention it is possible to increase production of autogenous lymphocytes for administration to the patient.

This application is a continuation-in-part of U.S. Patent applicationSer. No. 07/739,809 filed Aug. 2, 1991, now U.S. Pat. No. 5,206,008which is a continuation-in-part of U.S. Patent application Ser. No.07/685,078, filed Apr. 15, 1991.

FIELD OF THE INVENTION

The present invention provides an improved means for regulating theimmune response, for ameliorating effects of stress, and for avoidinguntoward effects of chemotherapy or exposure to irradiation byadministration of androstenediol (AED) and androstenetriol (AET). Theimproved means of regulating immune response can be utilized in treatinginfectious diseases and immune diseases such as diabetes and chronicfatigue syndrome, both diseases now considered to be immune responserelated syndromes.

BACKGROUND OF THE INVENTION

In vertebrates the development of host protection against pathogensrequires a selective host immune response that involves the mobilizationof the humoral and/or cellular mediated immune responses. Severalfactors adversely affect the body's protective response capability bycausing prolonged immuno-suppression or "down-regulation" of the immunesystem. It is, in reality, more appropriate to speak of "mal-regulation"or "deregulation" of the immune system than of "down-regulation" sincethe result is a failure to protect the body from assault.Immuno-suppression provides an opportunity for pathogens to grow in thehost. It does not matter what causes the primary insult to immunity. Theresulting inability to muster the appropriate immune response has thesame effect. Among the many different causes of immuno-suppression areviral, bacterial, fungal, yeast and parasitic infections, chemotherapy,irradiation, severe stress, chronic fatigue syndrome, diabetes mellitusand some forms of steroid therapy.

It has long been known that patients receiving steroid hormones ofadrenocortical origin at pharmacologically appropriate doses showincreased incidence of infectious disease. A. S. Fauci, immunolo.rev.65, 133-155 (1982); and J. E. Parillo and A. S. Fauci, Annual Review ofPharmacology and Toxicology 19, 179-201 (1979). Dehydroepiandrosterone,also known as 3-β-hydroxyandrost-5-en-17-one or dehydroiso-androsterone(referred to hereinafter as DHEA), is a 17-ketosteroid which isquantitatively one of the major adrenocortical steroid hormones found inmammals. M. E. Windholz, The Merck Index, Ninth Edition (1976); K. Diemand C. Lentner, Geigy Scientific Tables (1975). (Although DHEA appearsto serve as an intermediary in gonadal steroid synthesis, the primaryphysiological function of DHEA has not been fully understood. It hasbeen known, however, that levels of this hormone begin to decline in thesecond decade of life, reaching 5% of the original level in theelderly.)

Clinically, DHEA has been used systemically and/or topically fortreating patients suffering from psoriasis, gout, hyperlipemia, and inhas been administered to post-coronary patients. W. Regelson et al., NewYork Academy of Sciences 518, 260-273 (1988). In mammals DHEA has beenshown to have weight optimizing and anticarcinogenic effects.

DHEA has been used clinically in Europe in conjunction with estrogen asan agent to reverse menopausal symptoms and also has been used in thetreatment of manic depression, schizophrenia, and Alzheimer's disease.DHEA has also been used clinically at 40 mg/kg/day in the treatment ofadvanced cancer and multiple sclerosis. (Regelson, supra) Mildandrogenic effects, hirsutism, and increased libido were the sideeffects observed. These side effects can be overcome by monitoring thedose and/or by using analogues.

U.S. Pat. No. 5,077,284 entitled "Use of Dehydroepiandrosterone toImprove Immune Response" describes the subcutaneous or oraladministration of DHEA to improve the host's response to infections.U.S. Pat. No. 4,978,532 describes use of patch technology to deliverDHEA.

It is now disclosed that DHEA is a precursor in a metabolic pathwaywhich ultimately leads to more powerful agents that increase immuneresponse in mammals. That is, DHEA acts as a biphasic compound: it actsas an immuno-modulator when converted to androstenediol (5-androstene,3β,17β diol hereinafter referred to as βAED) or androstenetriol (5androstene 3β,7β,17β triol hereinafter referred to as βAET), but invitro has a certain lymphotoxic and suppressive effect on cellproliferation prior to its conversion to βAED and/or βAET. It is,therefore, now understood that administration of DHEA shows superiorimmunity enhancing properties as a result of its partial conversion to amore active metabolite.

An agent that would advance the protective regulation of the immunesystem without giving rise to undesirable side effects seen with DHEAadministration would provide particularly advantageous improvement ofhost resistance against infection. Protective regulation of the immunesystem could then be effected using lower doses of the chemotherapeuticagent, and would provide more immediate response with a wider range ofprotection.

DESCRIPTION OF THE INVENTION

The present invention provides compounds and compositions useful forenhancing the protective response of the immune system againstinfections. The medicinal compositions of the invention are also usefulfor treating other complications often accompanying immune suppression.The enhancement of the protective immune response may also be referredto herein as up-regulation or regulation of immune response.

The present invention provides means of synthesizing both α and β sterioisomers of AET. Both isomers are present in the body. However, since theactivity of the isomers varies, it is important that means of making theseparate isomers be provided. It is now known that the βAET is theactive agent in regulation of immune response. It is, however, probablethat the αAET, when in excess, is in equilibrium with βAET. However,because it is not known at this time what the response delay intervalis, the βAET is the composition that is the isomer used therapeutically.

Using βAED and βAET and protected analogues avoids certain side effectsresulting from use of DHEA. The use of βAED and βAET avoids many of theandrogenic side effects that occur when the precursor DHEA isadministered. With βAED and βAET it is possible to obtain rapid,controlled enhancement of immune response. Furthermore, as shown by theinformation below in Table 1, the βAED is unexpectedly superior to DHEAfor purposes of protecting against viral assault since the protectionoffered differs both quantitatively and qualitatively from protectioneffected by DHEA. The structures of βAED and the α and β isomers of AETare given below: ##STR1##

In addition to the medicinal compositions, the invention encompassesanalogues and derivatives of βAED and βAET. As indicated later in theexemplification of synthetic techniques, some of the substitutedcompounds are useful as starting materials or intermediates in makingβAED and βAET. βAED is available from commercial sources and has beenused as a raw material for production of other steroids. (See U.S. Pat.No. 2,521,586.) The product used in testing was obtained from Sigma.βAED and βAET may also be substituted with protective groups which, onhydrolysis yield βAED or βAET. Hence, acylated and alkylated derivativesare useful as precursors to the βAED and βAET. Compounds such as thoseof the formula: ##STR2## wherein R₁ may be H, alkenyl of 2-8 carbons,alkyl of 1-8 carbons, phenylalkyl of 1-4 carbons, phenyl or COR₂,wherein R₂ is H; alkyl of 1-8 carbons, alkenyl of 2-8 carbons,phenylalkyl wherein the alkyl has 1-4 carbons (including benzyl) orphenyl. Any phenyl moiety may have up to three substituents chosen fromamong hydroxy, carboxy of 1-4 carbons, halo, alkoxy of 1-4 carbons,alkyl of 1-4 carbons, or alkenyl of 2-4 carbons and wherein any alkylmay be a straight chain, branched chain, or the alkyl may be wholly orpartially cyclized.

The acute virus infection used as a model in the examples was chosenbecause of its widespread effects and the severity of the model to showprotection. The Coxsackievirus B4 is lethal to mice. In man, Coxsackie-viruses cause such varied pathologies as upper respiratory infection,pharyngitis, hemorrhagic conjunctivitis, meningitis, exanthem,encephalitis, hepatitis, infantile diarrhea, paralysis, pericarditis,and myocarditis. It is now believed that viruses of this group also havea role in the onset of juvenile diabetes

The use of βAED and βAET and protected analogues as taught hereinprovides high levels of protection to vertebrates, including humans,against morbidity arising from infections or exposure to immunesuppressive influences. In clinical medicine, treatment with βAED andβAET can lower morbidity in patients exposed to pathogenic organisms.These agents can be effectively used prophylactically in patients knownto be particularly susceptible to infection. Patients undergoing surgeryor chemotherapy or patients suffering from burns, hypoplastic oraplastic anemias, or diabetes are such susceptible patients who wouldbenefit from prophylactic administration of βAED and/or βAET. Also amongthe causes of immuno-suppression are viral, bacterial, fungal, yeast andparasitic infections, chemotherapy, irradiation, severe stress, chronicfatigue syndrome and some forms of steroid therapy. The compositions ofthe invention are particularly useful for treating patients sufferingfrom infections caused by viruses that destroy the body's immuneresponse, such as human immunodeficiency virus (HIV) and hepatitis.

Both βAED and βAET are most efficiently formulated using lipophiliccarriers such as DMSO. For subcutaneous administration to animals usedin the examples, the active agents were dissolved 1:1 DMSO/ethanol, thendiluted for subcutaneous administration to the animals. When thecompositions were administered by mouth, they were added to chow toprovide a composition containing 0.4% AED. For application to the skin,the AED may, for example, be dissolved in carrier material containingDMSO and alcohol, then applied to a patch. In such instances, the AED insolution may be added to another carrier such as glycerol beforeapplication of the 15 composition to the support material of the patch.For vaginal or rectal administration, the AED may be administered bysuppository, enema, or by application of creams, etc. Compositions ofthe invention may be administered by any method that will result incontact of the active agent with tissue of ectodermal origin. Suchmethods include subcutaneous or intradermal injection or topicalapplication. One means of topical application is the use of skin patchesimpregnated with the active agent. This means of delivery isadvantageous since it is non-invasive and easily administered byrelatively unskilled care providers. Compositions of the invention canalso be used in veterinary medicine to prevent morbidity that occursduring stress of shipping. Administration of βAED and βAET can beeffective as a means to prevent spread of infectious disease andintroduction of infectious organisms into the foods for humanconsumption. βAED and βAET can be administered by subcutaneousinjection, in food or drink, by patches applied to the skin, or byinhalation. A particular health concern is the spread of infectionthrough eggs. Eggs are frequently infected during development in thehen. Compositions containing active agents of the invention may be addedto the feed or water to prevent bacterial infection in the eggs.

When patches are used on animals or birds the skin should be exposeddirectly to the patch. When a patch is used, it may be necessary topluck or shave the bird or animal to expose the skin.

Other preferred methods of administration include buccal, sublingual,nasal or endotracheal routes. Sprays may be useful for this purpose. Fornasal administration, the active agent may be delivered as a powder thatis snorted. Inclusion complexes such as cyclodextrin inclusion complexesare appropriate compositions for administration to the oral-pharyngealand nasal mucosa.

βAED and βAET may also be given with vaccines to enhance immuneresponse. These agents may be administered either in a compositioncontaining the vaccine or may be given in a composition separate fromthe vaccine.

The compounds of the invention may also be administered to theintestinal mucosa by oral or rectal routes. Suppositories, solutions foruse as retention enemas, and creams or jellies are appropriate carriersfor use in rectal administration.

Compounds of the invention may be applied to the vaginal mucosa usingcreams, jellies, suppositories, or douching solutions. In order toenhance immune response at the site of exposure to infectious organisms,the compounds may be added to prophylactic vaginal preparations or maybe used as lubricants on condoms.

Administration of compositions of the invention has proven to be highlyeffective as a means of protecting against encephalitis and meningitis.The compositions of the invention may be administered intrathecallyeither at the spinal level or into the cisterna magna.

Active agents of the invention may be administered via ocular routeusing compositions in the form of drops, creams, or gel solutions orsuspensions adapted for ocular application.

βAED and βAET inhibit the adherence properties of body cells. Forpurposes of effecting the anti-adherence properties of the active agentsof the invention may be delivered directly to the epithelial tissueduring surgery. An example of such use would involve the application ofcompositions containing the active agents of the invention to theomentum in conditions such as infection, endometritis and malignanciesof the bowel and ovary wherein adherence of foreign cells or particlesto normal cells of the peritoneal lining is a problem. Compositions ofthe invention could, for example, be administered as mists or sprays.

It has now been shown in vivo that DHEA is converted to the βAED andboth isomers of AET. The βAED and βAET then act as regulators of theimmune response. In the skin, the conversion of DHEA to AED andsubsequently to AET appears to be one of the metabolic pathways of DHEA.The human skin has the enzymatic machinery to form 7β-OH DHEA and tocause 7β hydroxylation of AED to yield βAET, while the human adrenalcortex and liver can form only 7α-hydroxylation of DHEA, but not7β-hydroxylation. The following table indicates the metabolic pathwaysof DHEA.

    ______________________________________                                        Trivial or common name                                                                        systematic name                                               ______________________________________                                        (a) Dehydroepiandrosterone                                                                        (DHEA) 3β-Hydroxy-5-androsten-                                           17-one                                                    (b) Dehydroepiandrosterone                                                                        (DHEAS) 3β-Hydroxy 5                                     sulfate         androsten-17-one-sulfate                                  (c) 7 β-OH Dehydroepi-                                                                        --                                                           androsterone                                                              d)  Androstenediol  (AED) 5-androstene3β,17β,diol                   e)  Androstenetriol (AET) 5-androstene-3β,7β,17β,triol         (f)  --             7 keto-5- androstene-3β,17β, diol               (g)  --             5-androstene-3β,7α,17β,triol              (h)  --             5-androstene-3β, 17β, diol-3-sulfate            (i) Testosterone    17β-Hydroxy-4-androstene-3one                        (j) Androstenedione 4-androstene-3,17 dione                                   (k) Dihydrotestoserone                                                                            17β-Hydroxy-5 β androstane-3-one                (l) Androsterone    3β-Hydroxy-5β androstan-17-one                  ______________________________________                                    

DHEA (a) is known to have immune enhancing activity. However,dehydroepiandrosterone sulphate, "(b)", has been found to have noenhancing effect on the immune system. Both 5 androstene-3β, 11β,17β,-triol and 5 androstene-3β, 16β, 17β,-triol have immunosuppressiveeffects. Testosterone, "(i)", is known to have an effect on the hostimmune response, but this effect does not result in protection fromlethal infections. The qualitative and quantitative effects oftestosterone and the other sex hormones on the immune response are bothof a different nature and scope.

EXAMPLE 1

Use of androstenediol (5 androstene-3β, 17β, diol, βAED) results inmarked and significant resistance against viral and bacterial infection.Dose curve experiments were conducted in the following manner: βAED andDHEA were administered as a single depo dose of 8.3 mgs AED or 25 mgs.DHEA to SWR/J and C57BL/6J inbred mice. The mice were then challengedwith varying amounts of Coxsackievirus B4 (CB4) to determine protectivevalue of the active agents. βAED provided 50% protective dose againstcoxsackievirus B4, which was as much as 100 times greater thanprotection provided by DHEA. In addition, to the difference in ED50, thedegree of protection against virus mortality achieved withandrostenediol was also greater than the one obtained with DHEAinjection. The increased protection effected by βAED against virusinduced mortality was statistically significantly different that theprotection obtained by DHEA (P<0.05).

                  TABLE 1                                                         ______________________________________                                        DHEA AND AED IN SWR/J MICE MORTALITY PER                                      GROUP*                                                                        ______________________________________                                        CB4 10.sup.5                                                                           VIRUS ALONE 1/6                                                                              DHEA 0/6 AED 0/6                                      CB4 10.sup.6                                                                           VIRUS ALONE 3/6                                                                              DHEA 0/6 AED 0/6                                      CB4 10.sup.7                                                                           VIRUS ALONE 5/6                                                                              DHEA 1/6 AED 0/5                                      CB4 10.sup.8                                                                           VIRUS ALONE 4/6                                                                              DHEA 1/6 AED 0/6                                      ______________________________________                                         *No deaths occurred in control groups.                                        AED versus control pvalue = 0.0001.                                           DHEA versus control pvalue = 0.0017.                                          DHEA versus AED pvalue = 0.0588.                                         

As seen from these results, βAED is markedly more efficient than theprecursor DHEA in preventing mortality since an effective dose of βAEDwhich is 1/3 or less the dose necessary to obtain an effect with DHEA iseffective in achieving protection from mortality. A similar protectionfrom virus mediated mortality was also observed in the inbredC57BL/6J(b) strain. The two inbred mouse strains, the SWR/J and theC57BL/6J, differ in their major histocompatibility haplotypes, which areq and b respectively. These results show that up-regulation of theimmune response achieved with βAED in strains of differenthistocompatibility may be independent of the major histocompatibilitygenes on chromosome 17.

Recent reports show that the skin may have unique immune functions. J.W. Streilein and R. E. Tigelar, Photoimmunology, Parrish et al. eds.(Plenum Publishing, New York, 1983) pp. 95-130. Indeed the skin is knownto contain a population of cutaneous immune cells, which include theepidermal Langerhans cells and keratinocytes that produce a epidermalthymocyte-activating factor, similar to IL-1, in the murine system'sThy-1+ dendritic epidermal cell.

EXAMPLE 2

A composition containing 8 mg βAED was administered subcutaneously tooutbred ICR mice who were then challenged with a lethal dose ofStreptococcus faecalis strain X1515.OGlRF. Animals given βAED showedmarked resistance to morbidity as evident from reduction in mortalityfrom 57% in animals challenged with bacteria only, to 0% mortality inanimals infected and treated with a single subcutaneous (SC) dose ofβAED. Moreover, βAED above a certain threshold dose mediates aconsiderable proliferation of lymphocyte cells in the spleen and thymus,but only in infected animals. Administration of 8 mg/animal βAED withoutexposure to the virus did not cause proliferation. Histopathologicalexamination of the organs of inbred SWR/J mice infected with virus onlyand animals treated with βAED only, or βAED treated and virus infectedrevealed that βAED is effective in protecting from virus-inducedmyocardiopathy, and pancreopathy. Data presented in Table 2 below, showsthat βAED protects the host ICR inbred strain (Inst. of Cancer Researchstrain now supplied by Holland Sprague Dowley Company) from S. faecalis,but at a dose which is 1/3 the dose of DHEA required for the sameeffect.

                  TABLE 2                                                         ______________________________________                                        BAC-             DOSE              %                                          TERIUM  AGENT    mg/ANIMAL   24 hr.                                                                              MORTALITY                                  ______________________________________                                        S. faecalis                                                                           none     0           4/7   57                                         S. faecalis                                                                           DHEA     25          0/7   0                                          S. faecalis                                                                           AED      8.3         0/7   0                                          ______________________________________                                    

Protection was accomplished in an extremely acute infection where deathsoccurred within 24 hours (4/7 deaths in non-treated groups versus 0/7 inthe treated). Mice were challenged with a lethal dose of plasmidscontaining bacterium S. faecalis isolate X1515.OGlRF.

EXAMPLE 3

Wells containing monolayered urinary bladder tumor cells were coveredwith a 50 μmolar solution of either βAED or DHEA overnight before beingused in the assay. The results are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                               X1515.OG1RF                                                            WELL   DOSE        NO. ADHERED   % ADHERED                                    ______________________________________                                        Media  1.78 × 10.sup.6                                                                     1.77 × 10.sup.6                                                                       99.4                                         DMSO   1.78 × 10.sup.6                                                                     1.98 × 10.sup.6                                                                       111.2                                        DHEA   1.78 × 10.sup.6                                                                     1.01 × 10.sup.6                                                                       56.7                                         AED    1.78 × 10.sup.6                                                                     1.05 × 10.sup.6                                                                       59.0                                         ______________________________________                                    

As can be seen from Table 3, both DHEA and βAED inhibit the adherence ofthe bacteria to human urinary bladder tumor cells. It is believed thatβAED and AET have an effect on either membrane fluidity, or on acomponent which influences adhesion and/or penetration into the cell.Urinary Bladder Tumor cells (EJ6) were analyzed by flow cytometry usingthe fluorescent probe 1-4, trimethylammonio-phenyl-6 1,3,5 hexatriene(TMA-DPH) as a membrane probe to determine the effects of AED onmembrane fluidity.

Result

A. Samples 1 and 2 represent CONTROL cells grown in standard tissueculture media.

B. Samples 3 and 4 represent cells grown in the above control media with50 μM AED added.

    ______________________________________                                        Sample A 1.             0.121256                                              Sample A 2.             0.120548                                              CONTROL AVERAGE         0.120902                                              AED 50 μM 3.         0.135151                                              AED 50 μM 4.         0.142453                                              TEST AVERAGE            0.138802                                              ______________________________________                                    

The difference of 0.017899 between the control and βAED-treated cells issignificant and demonstrates that βAED caused a significant change incell membrane adhesion. It may be that an increase in cell membranerigidity (decrease fluidity) influences adherence.

EXAMPLE 4

Cell culture media was inoculated with 4×10⁷ Hela cells. DHEA was addedto a concentration 50 μM and βAED was added to a concentration of 50 μM.The cell culture exposed to DHEA showed a two-fold decrease in cellnumber. The cell culture exposed to the βAED, the control, and theculture exposed to the carrier alone showed a two fold increase or nochange in the number of viable cells. These observations indicate βAEDlacks deleterious effects of DHEA during the initial phase of cellresponse. Neither DHEA nor βAED had an effect on the number of virusinfectious particles in vitro.

EXAMPLE 5

Mice were injected with 8 mg βAED or 25 mg DHEA subcutaneously. The micewere then infected with Coxsackievirus B4, 10⁵ particles. The mice weresacrificed after 7 days. The spleen lymphocytes were removed andstimulated with Concavalin A, 5 μg/ml in vitro. Stimulation was measuredby ³ H-thymidine incorporation method. The βAED had a profound effect onthe proliferation of spleen lymphocytes with as much as 6.6 fold greaterproliferation evidenced. The data also indicates that, during theinitial phase following administration of the steroid DHEA results insome suppression of the immune system.

The results described above indicate clearly the advantage of using βAEDor βAET rather than DHEA to increase immune response to infections,since the initial immune suppression or toxicity that precedes theimmune up-regulation that is seen in treatment with DHEA is avoided.Hence, therapy with βAED and βAET, rather than DHEA is preferred toimprove immune response to infectious organisms.

The only change in structure between DHEA and AED is a reduction of theketo group at the 17 position to a β hydroxyl group. The AET which hasthe hydrogen at the 7 position replaced with a β hydroxyl group isproduced by a metabolic pathway in the skin. In other words, βAET is adownstream product of metabolic pathway from DHEA in the skin. The βAETappears to be the most effective compound for effecting immuneup-regulation and protection from untoward effects of stress,chemotherapy, and irradiation.

PREPARATORY METHOD #1

An important aspect of the invention is the preparation ofsterio-specific βAET for use as a medicinal. The synthesis wasaccomplished using the 7-oxo-3β, 17β acetoxyandrost-5-ene as a startingmaterial.

SYNTHESIS OF 3β,7β, 17β-TRIHYDROXYANDROST-5-ENE(I) and 3β, 7α,17β-TRIHYDROXYANDROST-5-ENE(II)

Chromic oxide oxidation of 3β,17β-diacetoxyandrost-5-ene L in glacialacetic acid gave 3β,17β-diacetoxyandrost-5en-7-one, (III), theintermediate to (I) or (II).

Aluminum isopropoxide reduction of (III) in isopropanol gave (I).lithium tri (sec-butyl) borohydride reduction of (III) intetrahydrofuran yielded (II).

PREPARATION OF 3β,17β-DIACETOXYANDROST-5-EN-7-0NE(III)

37.4 g(0.1 mol) 3β, 17β-diacetoxyandrost-5-ene (Steraloids A7850) in 400ml glacial acetic acid was reacted with 30.06 g (0.3 mol) chromium(VI)oxide (Aldrich 23,265-3) dissolved in 20 ml H20 and 20 ml glacial aceticacid. The CrO₃ solution was added drop-wise to the 3β,17β-diacetoxyandrost-5-ene solution while maintaining the temperature at55° C. for 4 hours. In order to decompose any unreacted CrO₃, methanolwas added to the reaction mixture followed by aqueous salt solution andether. Evaporation of the ether yielded 7.8 g(20% yield) of crudeIII,(details are given in lab book #1, pp. 10-16). Crystallization from95% EtOH yielded (III) m.p. 214°-215° C., DSC peak 191°-224° C., max at220° C. Lit.,¹ m.p. 218°-219° C., Lit.,² m.p. 224°- 225° C. (frommethanol).

Normal phase tlc: EtOAC-cyclohexane-EtOH (45:45:10), Rf=0.86. IR bands(cm -1: 1737,1666 (Lit.,² 1728,1668). ¹ H NMR (CDC13),(d), ppm:0.81(s,3H), 1.25 (s,3H), 2.02(s,6H), 2.25(m,H at C-4), 2.5(m,H atC-8),4.6(t,H at C-17),4.7(m,H at C-3), 5.72(s,1H); (Lit.,² 0.80(s,3H),1.20(s,3H), 2.03 (s,6H), 4.62(m,1H), 5.71(g,1H).) Reverse phase1c/ms(fast atom bombardment detection) detected m/z 389(M+HJ' ion in themajor 1c peak,(Lit.,2 m/z 388(M). Tentative C-13 nmr, (d), ppmassignments (CDC13) are:

    ______________________________________                                        Car- 1      2      3    4     5     6     7     8                             bon                                                                           ppm  38.35  35.74  72.04                                                                              43.03 164.22                                                                              126.44                                                                              201.18                                                                              65.82                         Car- 9      10     11   12    13    14    15    16                            bon                                                                           ppm  49.69  37.76  27.26                                                                              35.95 44.72  44.95                                                                              25.83 27.55                         Car- 17     18     19   20    21    22    23                                  bon                                                                           ppm  81.90  12.05  20.67                                                                              171.14                                                                              21.24 170.23                                                                              21.17                               ______________________________________                                    

PREPARATION OF 3β,7β,17β-TRIHYDROXYANDROST-5-ENE(I)

3β,17β-diacetoxyandrost-5-en-7-one was subjected to reduction byaluminum isopropoxide in isopropanol to give3β,7β,7β-trihydroxyandrost-5-ene.

PREPARATION OF 3β,7α,17β-TRIHYDROXYANDROST-5-ENE(II)

5.1 ml(5.1 mmol) lithium tri(seo-butyl)borohydride (AldrichL-Selectride) in tetrahydrofuran was rapidly added to 499 mg(1.28 mmol)of 3β,17β-diacetoxyandrost-5-en-7-one in 15 ml of freshly distilledtetrahydrofuran under nitrogen while stirring for 1.5 hours at ice-bathtemperature. 0.9 g KOH in 15 ml methanol was added, reaction mixturerefluxed for 0.5 hours, and then added 37.5 ml of 10% NaCl solution.After cooling in freezer(-20°), crystals formed which were filtered toyield 123.6 mg (19%) (11), m.p. 239°-45° C. Crystallization frommethanol yielded (II), m.p. 249.5°-253° C. ¹ H nmr (CD(OD)₃), (d) ,ppm:0.75(s,3H),1.01(s,3H), 3.1(m,1H), 3.6(t,1H),3.7(d,1H),5.50(d,1H).

Tentative C-13 nmr, (d),ppm assignments (CD(OD)3) are:

    ______________________________________                                        Carbon                                                                              1      2      3    4    5     6     7    8                              ppm   37.49  32.12  72.01                                                                              42.91                                                                              146.75                                                                              124.88                                                                              65.46                                                                              39.09                          Carbon                                                                              9      10     12   13   14    15                                        ppm   43.58  38.55  21.49                                                                              30.65                                                                               93.65                                                                               45.35                                                                              38.13                               Carbon                                                                              16     17     18   19                                                   ppm   42.57  82.30  11.57                                                                              19.53                                                ______________________________________                                    

DISCUSSION

Stereochemistry was assigned to 3β,7β, 17β-trihydroxyandrost-5ene(I) and3β,7α,17β-trihydroxyandrost-5-ene(II) by comparison withcholest-5-ene-3β,7β-diol and cholest-5-ene-3β,7α-diol proton nmr(Lit.3).L-Selectride reduction of (III) to produce (II) was carried out usingthe same reaction conditions given by Morisaki for the preparation of7α-hydroxycholesterol⁴. Carbon-13 resonances for stereoisomers (I) and(II) are shown. (Multiplicities (ppm))

    ______________________________________                                        ISOMER(II)              ISOMER(I)                                             ______________________________________                                        1        37.19          T     37.77                                           2        32.12          T     32.30                                           3        72.01          D     72.12                                           4        42.91          T     41.24                                           5        146.75         *     144.10                                          6        121.88         D     127.43                                          7        65.46          D     74.00                                           8        39.09          D     38.29                                           9        43.58          D     50.08                                           10       38.55          *     37.73                                           11       21.49          T     21.90                                           12       30.65          T     30.86                                           13       43.65          *     44.27                                           14       45.35          D     52.31                                           15       38.31          T     26.60                                           16       42.57          T     42.57                                           17       82.30          D     82.30                                           18       11.57          Q     11.57                                           19       19.53          Q     19.53                                           ______________________________________                                         Q = quartet, T = triplet, D = doublet and * = quaternary C                    (ISOMER II, 7OH axial)  (ISOMER I 7OH equatorial)                        

REFERENCES

1. Adolf Butenandt et al. Ber. 71B, 1316-22(1938).

2. Anthony J. Pearson et al., J. Chem. Soc. Perkin Trans. I,267-273(1985).

3. Leland L. Smith et al., J.Org.Chem.,Vol.38, No.1, 119-123 (1973).

4. Masuo Morisaki et al., Chem. Pharm. Bull. 35(5)1847-1852, (1987)

PREPARATORY METHOD #2

A second method of preparing the α and β isomers of AET has beendeveloped. The process uses, as a starting material, 3β, 17βdiacetoxyandrost-5-ene a commercially available reagent (StereloidsA7850). This compound is oxidized with chromium hexacarbonyl (Cr(Co)₆)to form 3β. 17β-diacetoxyandrost-5-en-7-one as described by Pearson(Pearson, et al., J. Chem Soc. Perkin. Trans. 1, 1985, 267.) The enoneformed was then reduced with triisobutylaluminum (TIBA) to give theacetylated 7α-hydroxy (3a) or 7β-hydroxy (3b) product depending on thesolvent used.

PREPARATION OF 3β, 7α. 17β-TRIHYDROXYANDOST-5-ENE

A solution of 3β,17β-diacetoxyandrost-5-en-7-one (0.4991 g., 1.285 mmol)and tetrahydrofuran (20 mL, dried over MgSO₄) were mixed under anitrogen atmosphere. TIBA (2 mL, 2mmol, in 1M toluene) was addeddropwise by syringe. The solution was stirred at room temperature forabout 7 hours. The reaction was terminated by the addition of ethylacetate (1.4 mL), the methanol (10 mL), and finally, by addition of 10mL of 50% acetic acid. This solution was added to 100 mL water andextracted with ethyl acetate (3 times with 50 mL.) The organic layerswere combined and then washed with saturated sodium bicarbonate solution(50 mL), saturated sodium chloride solution (2 times with 50 mL) andwater (50 mL). The organic layer was then dried over magnesium sulfateand the solvent removed by rotary evaporation to yield >96% crudeproduct. ¹ H NMR indicated that the crude product contained 3β,17β-diacetoxyandrost-5-en-7-one (starting material), 65% and3β,17β-diacetoxy-7α-hydroxyandrost-5-ene, 35%.

PREPARATION OF 3β,7β,17β-TRIHYDROXYANDROST-5-ENE

A solution of 3β,17β diacetoxyandrost-5-en-7-one (0.9581 g, 2.466 mmol)and pentane (30 mL), dried over MgSO₄) were mixed under a nitrogenatmosphere. TIBA (9.5 mL, 9.5 mmol, 1M in toluene) was then addeddropwise by syringe. The solution was stirred at room temperature forabout 1 hour. The reaction was terminated by the addition of dilutedhydrochloric acid (approximately 5mL). This solution was added to water(100 mL) and extracted with ethyl acetate (3 times with 50 mL). Theorganic layers were combined and then washed several times withsaturated sodium bicarbonate solution (50 ml), saturated sodium chloridesolution (two times with 50 mL,) and water (50 mL). The organic layerwas dried over magnesium sulfate and the solvent removed by rotaryevaporation to yield 86% crude product. ¹ H NMR indicated that the crudeproduct contained 3β,17β,-diacetoxy-7-β-hydroxyandrost-5-en-7-one (86%)and 3β,17β-diacetoxy- 7-α-hydroxyandrost-5-ene.

In both of the above instances, the final products were reduced inaccord with preparatory method #1.

The compositions of the invention containing βAED and βAET, are usefulin treatment any condition wherein a condition related to immuneresponse is present including diabetes mellitus, chronic fatiguesyndrome, stress, chemotherapy, and exposure to irradiation. One of themore depressing results of many of these conditions that involveaberrations of the immune response includes alopecia. The compounds ofthe invention are effective for treating the alopecia resulting fromsuch aberrations of immune response, including effects of chemotherapyand irradiation. In alopecia, the αAET should be of considerable benefitfor use in treatment. The compositions of both βAED and βAET are usefulin overcoming other untoward effects of immune suppression arising inthese conditions.

EXAMPLE 6

SWR/J inbred mice were injected with single depo doses of 0.5, 2.0, 4.0and 8.0 mg/animal of βAED, then challenged with 10⁷ PFU CoxsackievirusB4. The results are shown below:

    ______________________________________                                        AED dose, mg/animal                                                                           % cumulative survival                                         ______________________________________                                        0                17                                                           0.5              83                                                           2               100                                                           4               100                                                           8               100                                                           ______________________________________                                    

Based on these results, the theoretical dose (extrapolated) of βAEDnecessary to achieve 50% protection from infection with 10⁷ PFU of CB4is 0.25 mg/animal.

The example demonstrates that βAED is 50 to 100 times more potent thanDHEA for protection from Coxsackievirus B4.

EXAMPLE 7

In an attempt to evaluate effect of βAED on pathologies of the heart andpancreas in infected mammals, three groups of inbred SwR/J mice werecompared. The groups studied included animals infected with virus only,animals treated with βAED only, and animals infected with virus andprotected with βAED. The comparison revealed the following:

    ______________________________________                                                 Heart          Pancreas                                              ______________________________________                                        AED        unremarkable     unremarkable                                      VIRUS      focal areas of multiple                                                                        severe necrosis                                              necrosis with substantial                                                                      (Pancreopathy)                                               myocardial calcification.                                          AED PLUS   no evidence of   no evidence of                                    virus      induced myocardiopathy                                                                         virus induced                                                                 pancreopathy                                      ______________________________________                                    

These results demonstrate that βAED given as a single depo dose at 8.0mg/mouse protected the heart tissue from virus induced myocardiopathy,and also protected the pancreas from virus induced necrosis of thisorgan. These results show that βAED can be used effectively in theprotection from virus-induced cardiovascular and pancreatic disease, inparticular myocardiopathies and pancreopathies. Previously there were noeffective drugs to protect these organs from virus induced damage.

EXAMPLE 8

Effectiveness of βAED, αAET, and βAET were compared by challenging micewith Coxsackievirus B4, 5.0×10⁷ pfu/animal. The following datarepresents total cumulative mortality (n/6) at indicated days postinfection with virus.

    ______________________________________                                                  Cumulative mortality                                                          (Days Post Infection)                                               Treatment Group                                                                           2      4     6    8   10   12  14   16                            ______________________________________                                        Virus alone 0      1     6    *   *    *   *    *                             βAED .5 mg/virus                                                                     0      0     1    1   6    *   *    *                             αAET .5 mg/virus                                                                    0      1     4    4   4    4   4    4                             βAET .5 mg/virus                                                                     0      0     1    1   2    2   4    4                             ______________________________________                                    

While the αAET appeared to protect two animals from the virus, theinitial mortality would indicate that at least in early stages, there islittle or no protection from the αAET. Other studies indicate that thereis far less protection with αAET than with either βAED or βAET. Clearly,the βAET is the most effective agent for both early and longer termprotection. It appears that there is a limited protection attributableto αAET. Therefore, this isomer would not be as beneficial as βAET foreffecting enhancement of immune response.

The use of βAED or βAET as active agents to provide regulation of theimmune system makes it possible to effectively regulate the host immuneresponse against viral, bacterial and other infections. In the case ofvirus-induced heart or pancreatic infection where no other antiviralchemotherapeutic modality exists βAED and βAET have value asprophylactic protective agents. The protective value of βAED and βAET isparticularly important to patients undergoing surgical procedures orsuffering injuries where resistant strains of organisms such aspseudomonas present a serious threat. Examples of such patients arethose undergoing bowel surgery or suffering from gunshot wounds of theabdomen. Patients with history of conditions such as rheumatic feverwould also benefit from prophylactic use of βAED and βAET. The mode ofadministration in a particular case will depend on the infectious agentagainst which protection is sought and the target tissue of theorganism. While the administration subcutaneously as a depo is effectiveagainst systemic infections as shown by the data presented above, whenthe compositions are given to assist the body in meeting an infection ina particular tissue, it may be advantageous to administer the activeagents to the tissues most affected.

The carrier used in a given instance will depend on the mode ofadministration. Both AED and AET are lipophilic compounds. They are moresoluble than DHEA in water. Solvents for lipophilic steroids are knownin the art and would be used as carriers for these compounds. Examplesof such carriers are glycols such as polypropylene glycol, polyethyleneglycol and cyclodextrins, especially the intrinsically amorphouscyclodextrins. Other vehicles that should be considered include fattyacid esters of polyoxyethylene sorbatan (Tweens) or sorbitan (Spans) toprepare oil-in-water emulsions.

EXAMPLE 9

Capsules of a formulation of AED for oral administration are prepared bycontaining 15 mg. βAED, 150 mg. starch, and 5 mg. magnesium stearate.The capsules are administered daily or twice a day to achieve a dailydosage of 15 mg. per day.

In the laboratory, βAED was added to the chow of the animals at a rateof 0.4% of the diet. When the animals who had been fed the dietcontaining βAED were exposed to Coxsackievirus B4 by injection in accordwith the teachings of Example 1, the animals who had been fed the βAEDsurvived, while control animals all died.

EXAMPLE 10

A preparation for application to the skin or mucosa may be prepared inthe following manner:

    ______________________________________                                        Ingredient        % w/w                                                       ______________________________________                                        AED               0.5%                                                        glyceryl monostearate                                                                           3.0%                                                        propylene glycol  13.0%                                                       Petrolatum        83.5%                                                       ______________________________________                                    

When βAED or βAET or their analogues are administered orally, the activeagents may be utilized more efficiently if the active agents areprotected from destruction and absorption in the upper gastro-intestinaltract. The active agents are most effective when the period of exposureto the mucosa of the intestinal tract is increased. Hence use ofcapsules containing the active agents in formulations that effect slowrelease in the intestine are appropriate for treatment of intestinaldisorders such as Crohn's disease and colitis. Use of retention enemasfor treatment of inflammation of the large bowel is also appropriate.

A formulation for administration as a retention enema may be formulatedin the following manner:

EXAMPLE 11

    ______________________________________                                        Ingredient       w/w %                                                        ______________________________________                                        βAET         4%                                                          Propylene glycol 96%                                                          ______________________________________                                    

When the active agent is administered to the mucosa of the oral cavity,it may be administered as a buccal tablet or spray for use in theoral-pharyngeal cavity and the nasal cavities.

The compositions could also be administered to the bronchial tree viainhalation. This means of administration would be particularly useful intreating patients with lung infections or in treating other lungconditions such as black lung disease or emphysema that often arecomplicated by opportunistic infections. The compositions could be givenby aerosol into the trachea or administered in mist along with otheragents used in respiration therapy.

The administration of the βAED and βAET to the skin can be accomplishedusing patches wherein a support is impregnated with the active agent orusing implants that provide slow release of the active agents.

Patches for the administration of AET or AED can be formulated asadhesive patches containing the drug. For example, the patch may be adiscoid in which a pressure-sensitive silicone adhesive matrixcontaining the active agent may be covered with a non-permeable backing.The discoid may either contain the active agent in the adhesive or mayhave attached thereto a support made of material such as polyurethanefoam or gauze that will hold the active agent. Before use, the materialcontaining the active agent would be covered to protect the patch.

EXAMPLE 12

A patch composed of trilaminate of an adhesive matrix sandwiched betweena non-permeable backing and a protective covering layer is prepared inthe following manner:

To a pressure-sensitive silicone adhesive composition BIOPSA™ Q7-2920(Dow Corning Corp., Midland, Michigan, U.S.A.) in cyclohexane (50% w/v)is added sufficient βAED to provide a 0.5% βAED composition. Theadhesive is applied to a polyester film to provide in successive layersto provide about 2 mg of active agent per cm². The film containing theadhesive is then made into patches of 10 cm². Patches would be coveredwith a protective layer to be removed before application of the patch.Patches may be prepared containing permeation enhancers such ascyclodextrin, butylated hydroxyanisole, or butylated hydroxytoluene.However, it should be remembered that the active agents of thisinvention are effective on application to the epidermal tissue. When thepatches are to be applied to thin or abraded skin, there is little needto add a permeation enhancer.

Compositions of the invention can be administered as a prophylacticduring radiation therapy or chemotherapy or after exposure toirradiation whether the exposure occurs as a result of environmentalaccident or therapy. Other instances when use of these immuneup-regulators would be appropriate is in treatment of burns, hypoplasticand aplastic anemias, diabetes, and in the elderly during epidemics.Their use is also beneficial in preventing or mitigating effects ofexposure to dangerous infectious organisms, as was demonstrated by thedata related to cardiopathies and pancreopathies. Such use isparticularly indicated in populations exposed to organisms that targetthe immune system, such as HIV infections. In certain instances thecompositions taught herein can also be used as immune modulators in theproduction of blocking antibodies to counteract hypersensitivityreactions.

As indicated previously, patients scheduled to undergo bowel surgery orother "dirty" surgical procedures could receive a dose of βAED or βAETprophylactically. Use of the compositions as taught herein beforeinvasive dental procedures or oral surgery should be considered.

As indicated in the tables and previous discussion, the compositions ofthe invention can be used to prevent adhesion of bacteria to thetissues. The prevention of cell to cell adhesion resulting fromadministration of the compositions also has applications for preventionof thrombosis. Compositions of the invention can be administered as aslow drip into blood vessels when prevention of formation of a thrombusis necessary. An example of such use would be a drip into an arteryfollowing thrombolectomy or for prevention of cerebral thrombosis.Instillation into the bladder could also be beneficial for prevention ortreatment of urinary infection. The administration of the βAED or βAETcan be employed as a means of preventing formation of infectious foci.

βAED and βAET are both effective in overcoming effects of ultravioletdepravation. Hence, administration of the compositions to overcome theeffects of depression related to light deprivation (usually calledseasonal adaptive disorder) is appropriate.

βAED and βAET may be used as adjuncts in vaccination to increaseresponse to an immunogen. Not only will these agents increase responseto the vaccine, they will also increase ability to protect againstdisease before the body has responded with increase in specificantibodies. Such use is particularly appropriate in instances whereinhibition of immune response can be a complicating factor as is thecase in patients suffering from, for example, malignancies, AIDS, orenvironmental factors such as exposure to pesticides. It is, of course,understood that use as adjunct to vaccination would be appropriate invertebrates other than man, including vaccination of pets, dairyanimals, meat-producing animals, fish, and chickens. Chickens areparticularly prone to develop infectious diseases when living inconfined conditions. Coccidiosis, Salmonella infections, viralinfections, including those giving rise to malignancies such as leukemiaand sarcoma (caused by a retrovirus) are particularly common amongchickens grown under modern commercial conditions. The active agents ofthe invention may be given by any means that results in contact of theagent with tissue of ectodermal origin.

The effect of Coxsackievirus on humans has been noted previously. Thevalue of avoiding such effects, especially in children, is clear.Effects of other viruses such as chickenpox-herpes zoster is consideredan important cause of debilitating illness in the elderly. Furthermore,chickenpox in susceptible adults often causes severe illness. Inchildren, chicken pox can cause death when the child is subjected toimmuno suppressive therapy or is genetically immune deficient. βAED andβAET a useful for prophylatic treatment of susceptible persons who havebeen exposed to infection. Finally, the protection of the fetus andnewborn from HSV infection is a very important application of theinvention. βAED and βAET can be administered during the third trimesterto HSV-infected women as a means of protecting the newborn.

In vitro, these compounds can be used in commercial setting to inducelymphocyte proliferation. The use of βAED and βAET would increase yieldof products of such proliferation in tissue culture. In the clinicalsetting, βAED and βAET can be given to effectively enhance patients'ability to combat infections. Patient lymphocytes may be withdrawn,reproduced in vitro in media containing βAED or βAET to increaseproliferation of lymphocytes, and the lymphocytes so primed for responsecould then be reintroduced into the patient. In cases such as malignancyor other cellular disease, the malfunctioning cells can be inactivatedby known means before proliferation in growth media.

The compositions of the invention may also be used prophylactically toprotect animals from the consequences of infection by pathogenicorganisms. It is known that under the stress of shipment to marketanimals often become susceptible to infections that are not ordinarilyserious, but that can cause the animals to loss much weight en route tothe packing house. Such loss may be avoided by administration βAED andβAET and analogues disclosed herein. The active agents can be given bypatch, injection, or in feed. Because the active agents are mosteffective when the period of exposure to the tissue of ectodermal originis extended, when the active agents are administered through the GItract, compositions should be modified to extend the period of exposureof the active agent to the intestinal mucosa and to protect the agentsfrom destruction in the upper GI tract. Hence, use of capsules thateffect slow release in the intestine is appropriate. The capsules may beplaced in baits for administration to animals. To treat infections ofthe large bowel, the active agents may be given by retention enema.

βAED and βAET may be administered to the mucosa of oral, pharyngeal, andnasal cavity by tablet, a lozenge, by administration as a spray for usein the oral-pharyngeal cavity, or as a nasal spray.

Administration to the skin may be accomplished using patches wherein asupport to be applied to the skin is impregnated with the active agent.If the host is a mammal or bird, it may be necessary to shave or pluckthe region to which the patch is applied.

A preferred method of administration is by subcutaneous injection as adepo. The method is particularly appropriate for administration of theactive agents to mammals, since subcutaneous injection is easilyperformed and the effect is relatively long lasting.

βAED and βAET are already present in the body as natural components.They do not pose a serious toxic problem at levels known to be safe;they appear to be chemically quite stable.

The dosages used will depend on the size and condition of the host. Testdata indicated in this application was obtained in small animals. Inlarger adult mammals daily dosage of 0.2 to 30 mg/da. of AED is apreferred dosage. For AET the preferred dosage is usually in the rangeof 0.001 to 20 mg/da, with 0.001 to 1 mg/da. being the more preferreddosage. However, the dosage will vary depending on the route ofadministration. Subcutaneous, inhalation and intrathecal administrationare methods that would require lower dosages of the active agents.

It is, of course, understood that analogues of βAED and βAET havingprotective groups can be administered to the host as a means ofdelivering βAED or βAET to target tissues. Acylation is a preferredmethod of protecting the compounds. Acylated compounds wherein R₁ isCOR₂ are also appropriate compounds for use as starting material fromwhich to make βAED and βAET.

The active agents, βAED and βAET, can be given in conjunction with otheractive agents which may be given simultaneously or may be incorporatedin compositions containing βAED or βAET. βAED and βAET can be given withanti-infective agents such as antibiotics, antiviral agents,anti-fungals, antiparasitic agent to potentiate the activity of thesedrugs by up-regulating protective immune response. Antiviral agentsinclude, for example, Dideoxyinosine, AZT, acyclovir, etc. Other activeagents that may be combined with the AED and AET include antiallergicmedications such as epinephrine.

Finally, medicinal compositions containing βAED and βAET areparticularly valuable for use in combating viral infections in patientswho have suffered from infections exacerbated by immuno-suppressivetherapy. One of the major complications in patients with tissuetransplants is the opportunistic infection with viruses that ordinarilydo not cause serious disease symptoms. Use of the compositions of theinvention, which result in rapid protective regulation of the immuneresponse, allows the medical team to place the patient on "see-saw"therapy to avoid transplant rejection while regulating the immuneresponse to avoid overwhelming infection.

We claim:
 1. A composition of matter comprising a growth media capable of supporting lymphocyte cell growth in vitro containing, as a lymphocyte proliferation-enhancing agent, a lymphocyte proliferation enhancing amount of a compound of the formula: ##STR3## wherein R₁ may be H, alkenyl of 2-8 carbons, alkyl of 1-8 carbons, phenylalkyl wherein the alkyl is of 1-4 carbons, phenyl or COR₂, wherein R₂ is H, alkyl of 1-8 carbons, alkenyl of 2-8 carbons, phenylalkyl wherein the alkyl has 1-4 carbons or phenyl and wherein any phenyl moiety may have up to three substituents chosen from among hydroxy, carboxy of 1-4 carbons, halo, alkoxy of 1-4 carbons, alkyl of 1-4 carbons, or alkenyl of 2-4 carbons and wherein any alkyl may be a straight chain, branched chain, or the alkyl may be wholly or partially cyclized.
 2. A composition of claim 1 wherein R₁ in all instances is H.
 3. A composition of claim 1 wherein the lymphocyte proliferation-enhancing agent is 5-androstene-3β,17β diol.
 4. A composition of claim 1 further comprising lymphocytes.
 5. A composition of claim 4 wherein the lymphocytes are of human origin.
 6. A composition of claim 1 wherein the lymphocyte proliferation-enhancing agent is 5-androstene-3β,7β,17β triol.
 7. A method of increasing lymphocyte proliferation in vitro by providing, in a growth media containing lymphocytes, a lymphocyte proliferation enhancing amount of at least one of 5-androstene 3β,17β diol and 5-androstene 3β,7β,17β triol.
 8. A method of increasing lymphocytes of autogenous origin in a patient, the method comprising:1. Removing blood from said patient and separating lymphocytes from the blood of said patient;
 2. Growing lymphocytes obtained in step (1) in a media containing at least one of 5-androstene 3β,17β diol and 5-androstene 3β,7β,17β triol; and
 3. Separating lymphocytes from the growth media and administering lymphocytes obtained from the growth media to the patient from whom the lymphocytes were obtained in step
 1. 